During the 2002 and 2003 transmission seasons the United States experienced large epidemics of fever and encephalitis caused by West Nile virus (WNV), with more than 13,000 cases of human disease and almost 500 deaths reported to the CDC. Significant morbidity and mortality was also reported in equines and many avian species. Currently there are no vaccines or antiviral therapies to treat or prevent WNV infection in humans. In addition, no specific determinants of WNV virulence have been identified that might be exploited as targets for antiviral measures or incorporated into a recombinant, live attenuated WNV vaccine. The identification of specific markers for attenuation of WNV is critical to the timely production of attenuated vaccine candidates, and will provide more detailed insights into aspects of the basic biology of WNV that are currently not well defined. The envelope (E) protein of flaviviruses is the major virion surface protein and mediates two critical functions of viral infectivity - binding to target cell receptor(s), and fusion with target cell membranes. Attenuation of many flaviviruses, including attenuated vaccine strains of yellow fever and Japanese encephalitis viruses, is mediated at least in part by mutations in the E protein that affect either or both of these two essential functions. Structural and functional analyses have indicated that receptor binding and membrane fusion are mediated via distinct structural subunits of the E protein. The objective of this proposal is to derive variants of WNV with an attenuated growth and/or virulence phenotype by specific mutation of putative functional domains/motifs within the E protein of an infectious clone derived from a virulent North American strain of WNV. This will be achieved via two specific aims: 1. Mutation of membrane fusion associated structures; 2. Mutation of conserved, surface exposed residues within the putative E protein receptor binding domain. These studies will identify E protein residues that can impart an attenuated phenotype and these will be exploited for WNV vaccine development in a subsequent R01 grant application.